Renal cell carcinoma (RCC) is a highly prevalent malignancy with a high mortality rate. Sporadic and germline mutations and/or loss of the von Hippel-Lindau (VHL) tumor suppressor gene have been linked to varying levels of risk for RCC. VHL disease patients have one wild type VHL allele and one inactivated/mutated VHL allele. Tumor formation occurs when the normal copy of VHL is lost or inactivated. Given that VHL 2B missense mutation confers a high risk for RCC, the proposed project aims to identify key molecular changes that occur in renal cells following VHL inactivation that can provide insight into RCC tumor formation. It is well understood that functional pVHL protein under normal oxygen conditions targets hypoxia inducible factors (HIF) for proteosomal degradation, while under low oxygen conditions or mutated VHL allele, VHL fails to regulate HIF-1 subunits. HIF-1 subunits dimerize with HIF-2 subunits, translocate to the nucleus and are responsible for transcriptional activation of genes. Some HIF gene targets have been identified; most notably those important for angiogenesis and glucose metabolism, which are normally upregulated in RCC and other malignant tumors. The focus of this project is to identify a HIF gene target profile by comparing levels of gene expression when HIF-11 and/or HIF-21 are differentially regulated by VHL status using transgenic mouse models. To better understand the genetic changes associated with RCC tumor formation, primary epithelial kidney cells from newborn conditional Vhl null and Vhl 2B transgenic mouse lines will be used and will be harvested and minimally manipulated in an ex vivo system. After which, these cells will be reintroduced to their native environment, renal capsules of immunocompromised mice, to study their tumorigenic potential. The cellular composition of the generated pooled cell lines wilI be identified to investigate which cell type is most at risk for developing tumorigenesis. Whether renal carcinoma is due to VHL disease or sporadic mutations, this study bears great weight to the public, as it is imperative that we understand what changes occur in kidney cells to become tumorigenic. The proposed research will identify the most at-risk cell population and analyze the molecular changes in this at-risk cell population that result in tumor growth as well as identify a HIF gene target profile for these changes in order to have greater insight into RCC tumorigenesis.